Identification, distribution, and expression of novel genes in 10 clinical isolates of nontypeable Haemophilus influenzae

Abstract

We hypothesize that Haemophilus influenzae, as a species, possesses a much greater number of genes than that found in any single H. influenzae genome. This supragenome is distributed throughout naturally occurring infectious populations, and new strains arise through autocompetence and autotransformation systems. The effect is that H. influenzae populations can readily adapt to environmental stressors. The supragenome hypothesis predicts that significant differences exist between and among the genomes of individual infectious strains of nontypeable H. influenzae (NTHi). To test this prediction, we obtained 10 low-passage NTHi clinical isolates from the middle ear effusions of patients with chronic otitis media. DNA sequencing was performed with 771 clones chosen at random from a pooled genomic library. Homology searching demonstrated that approximately 10% of these clones were novel compared to the H. influenzae Rd KW20 genome, and most of them did not match any DNA sequence in GenBank. Amino acid homology searches using hypothetical translations of the open reading frames revealed homologies to a variety of proteins, including bacterial virulence factors not previously identified in the NTHi isolates. The distribution and expression of 53 of these genes among the 10 strains were determined by PCR- and reverse transcription PCR-based analyses. These unique genes were nonuniformly distributed among the 10 isolates, and transcription of these genes in planktonic cultures was detected in 50% (177 of 352) of the occurrences. All of the novel sequences were transcribed in one or more of the NTHi isolates. Seventeen percent (9 of 53) of the novel genes were identified in all 10 NTHi strains, with each of the remaining 44 being present in only a subset of the strains. These genic distribution analyses were more effective as a strain discrimination tool than either multilocus sequence typing or 23S ribosomal gene typing methods.

FIG. 1.

Distribution of randomly selected library clones with respect to the H. influenzae Rd KW20 genome. A map of the Rd chromosome was divided into sections, each representing 0.15 Mb. The number of clones demonstrating homology to each Rd section is noted, for a total of 684 Rd-like clones. The locations of 21 clones that contained both Rd and contiguous non-Rd sequences are shown in parentheses. Asterisks represent the positions of the six rRNA loci on the Rd genome (22). The 15 clones that contained rRNA sequence were not included in the figure, as it was not possible to determine which of the six regions was represented in each of these clones.

FIG. 2.

Phylogenetic tree generated using sequences from all 187 STs listed in the MLST database and showing the relative distances among the 10 clinical strains of H. influenzae (indicated to the right) characterized in the present study.